To determine whether HIV-1 p24 antigen would improve transfusion safety, more than 500,000 donations were tested at 13 U.S. blood centers using test kits from two manufacturers. Units found repeatedly reactive were retested and confirmed by neutralization. A subset was tested for HIV DNA by PCR. Selected donors with confirmed and non-confirmed HIV-AG were contacted to identify risk factors and for retesting of HIV markers. HIV-AG was confirmed in five donors (0.001 percent of all donations tested), all of whom were ELISA and PCR-positive. 3 of these had other infectious diseases and 2 of them risk factors that would have led to self-exclusion. None of the non-neutralizable, Ag positive samples were confirmed by PCR or virus culture. The results of this study suggests the rarity of antigen-positive, antibody negative samples in blood donations, as well as the lack of significance of HIV-AG testing to the safety of the nations blood supply.